Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

Selective promotion of ferrous ion-dependent lipid peroxidation in Ehrlich ascites tumor cells by histidine as compared with other amino acids.

Among tumors in general, Ehrlich ascites tumor cells are particularly resistant to lipid peroxidation. In this study lipid peroxidation was measured in terms of the formation of malondialdehyde-equivalent material in Ehrlich tumor cells during incubation in vitro. It was shown that the high antioxidant potential of these cells could be overcome by a strong radical-promoting agent like ferrous ion. Various amino acids were tested for their capability to augment the effect of Fe(II). Histidine and its 3-methyl-derivative turned out to be the most effective pro-oxidants, whose action could be ascribed to the presence of the imidazole group. From studies with homogenized and denatured cells it was concluded that lipid peroxidation stimulated by Fe(II)-histidinate is an autoxidation process and that no carrier effect of iron by histidine is predominating. The stimulatory action of Fe(II)-histidinate could be completely suppressed by vitamin C, which was shown to be a potent anti-oxidant under the conditions used. The combined application of Fe(II)-histidinate and vitamin C may offer a means to study lipid peroxidation of Ehrlich tumor cells in a controlled manner.

Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells.

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.

Detection of 4-hydroxynonenal as a product of lipid peroxidation in native Ehrlich ascites tumor cells.

4-Hydroxynonenal, which is a major product of lipid peroxidation in rat liver microsomes, was detected in native Ehrlich ascites tumor cells. Its formation was stimulated either by ferrous ions or by Fe(II)-histidinate. The identification was based on chromatographic (TLC/HPLC) and ultraviolet-spectroscopic evidence using synthetic 4-hydroxynonenal as reference. Highest values of 4-hydroxynonenal concentration (about 0.1 microM in the cell suspension) after 30 min of incubation were observed with Fe(II)-histidinate as stimulant. Saturation was already reached after an incubation period of 10 min. The results confirm the expectation by Schauenstein and Esterbauer (in Submolecular Biology and Cancer, Ciba Foundation Series 67 (1979) pp. 225-244, Excerpta Medica, Amsterdam) that endogenous lipid peroxidation gives rise to a distinct intracellular level of alpha, beta-unsaturated aldehydes. A simple hypothetical mechanism for the formation of 4-hydroxynonenal from n-6-polyunsaturated fatty acids is presented.

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions. Therefore, this method was used to subsequently compare the effectiveness of MPO inhibitors that block production of HOCl with compounds that act as HOCl traps. The efficiency of MPO inhibitors to prevent LDL damage increased in the series benzohydroxamic acid < salicylhydroxamic acid < 3-amino-1,2,4-triazole < sodium azide < potassium cyanide < p-hydroxy-benzoic acid hydrazide, while for the HOCl traps the protective efficiency increased in the series glycine < taurine < methionine. We conclude that HOCl traps may have high potential therapeutic impact in vivo due to their low toxicity, although high concentrations of them would have to reach sites of inflammation. In contrast, only low concentrations of a specific MPO inhibitor would be required to irreversibly inhibit the enzyme.

Basic aspects of the biochemical reactivity of 4-hydroxynonenal.

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups. Among the primary reactants for HNE are the amino acids cysteine, histidine and lysine, which--either free or protein-bound--undergo readily Michael additions to the C=C bond. After this primary reaction, which confers rotational freedom to the C2-C3 bond, secondary reactions may occur involving the carbonyl and the hydroxy group. Primary amines may alternatively react with the carbonyl group to form Schiff bases. Reactions which do not fit into this scheme are the oxidation and the reduction respective of the carbonyl group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed.

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.

Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic activity, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.

Mutual dependence of growth modifying effects of 4-hydroxynonenal and fetal calf serum in vitro.

Recently, the hypothesis has been put forward that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, contributes to the mechanisms of oxygen toxicity and to the selective pressure exerted by exposure to hyperoxia. Here it has been studied whether HNE itself is involved in mechanisms that convey increased resistance of the cells to the toxicity of HNE. The following four cell lines, different in their basic biological features, were used: nonmalignant Chinese hamster lung fibroblasts V79 (established cell line), human carcinoma HeLa (established cell line), pigmented murine melanoma B16f10 (primary culture), and amelanotic murine melanoma B16BL6 (primary culture). The cells were pretreated in vitro with a toxic dose of HNE (50 microM), and afterwards the effect of a second exposure to the same dose of HNE on 3H-thymidine incorporation was examined. Cells were cultured in the absence and in the presence of fetal calf serum (FCS), because it had been shown that a growth modifying effect of HNE depends on an unknown serum factor. The results showed that, regardless of the type of cells, preculturing them with 50 microM HNE in the presence of serum changed the reactivity of the cells to added serum as well as to additional HNE treatment. Thus, HNE precultured cells incorporated less 3H-thymidine in the presence of serum than if cultured under serum-free conditions. On the other hand, HNE precultured cells became less sensitive to further HNE treatment, but only if cultured in the presence of serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS.

A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated.

4-Hydroxynonenal modifies the effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of HeLa carcinoma cells.

In this study, the effect of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-poly-unsaturated fatty acids, on the expression of the c-fos proto-oncogene and growth factor-induced proliferation of HeLa carcinoma cells in vitro was investigated. The Fos protein forms the heterodimer AP-1 with the Jun protein and regulates the cell cycle by inducing cyclin D1. Agents that are able to induce c-fos include serum, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), all of which were used in this study. The proliferation rate was determined by cell counting (viable and dead cells according to trypan blue exclusion) and the BrdU assay. The c-fos mRNA level was monitored by the reverse transcriptase/polymerase chain reaction. In the absence of HNE, serum-deprived cells responded to serum stimulation with a more than 10-fold increase of the c-fos mRNA level as well as with an increased rate of DNA synthesis and cell multiplication. Both EGF and PDGF (applied in combination with insulin) were able to substitute for FCS and induced rapid growth of the tumor cells preincubated in serum-deprived medium. In the absence of growth factors a negative correlation between the HNE concentration (range: 1-250 microM) and the c-fos mRNA level was observed. We suppose that HNE interferes in this case with the basal activity of the c-fos promoter. EGF, when applied after the HNE treatment, induced rapid growth of the tumor cells preincubated in serum-free medium, if HNE was used in a physiological concentration (1 microM). No difference was observed compared to the HNE-free control. c-fos mRNA level was nearly unchanged. In contrast, a cytotoxic concentration of the aldehyde (100 microM) caused a complete inhibition of proliferation, although a twofold increase of the c-fos mRNA level immediately after the aldehyde treatment was observed. A similar effect of HNE in cytotoxic concentration on c-fos expression was observed when cells were grown in presence of PDGF instead of EGF. Hence, in both cases HNE possibly interferes with the signal transduction pathway, which is initiated by external growth factors. The increased c-fos expression might be part of an abortive attempt to overcome the stressful condition raised by a cytotoxic concentration of HNE.

Hypochlorite induces the formation of LDL(-), a potentially atherogenic low density lipoprotein subspecies.

Oxidation of low density lipoprotein (LDL) induced by hypochlorous acid (HOCl) leading to LDL(-), a minimally oxidized subspecies of LDL, was investigated. LDL(-) is characterized by its greater electronegativity and oxidative status, and is found in plasma in vivo. Its concentration was found to be elevated under conditions that predispose humans to atherosclerosis. We found that HOCl also converts LDL rapidly to an even more oxidized state, identified as LDL(2-), which is more electronegative than LDL(-). After milder oxidation for short durations, formation of LDL(-) takes place while less LDL(2-) is formed. Under these conditions, addition of methionine not only suppressed further oxidation of LDL but also favored the formation of LDL(-) over LDL(2-), possibly by removing chloramines at lysyl residues of LDL. The presence of lipoprotein-deficient plasma did not prevent HOCl-mediated conversion of LDL to more electronegative species. It is concluded that the HOCl-mediated conversion of LDL into more electronegative species might be physiologically relevant.

Influence of age on the release of reactive oxygen species by phagocytes as measured by a whole blood chemiluminescence assay.

Polymorphonuclear and mononuclear phagocytes play an important role in host defense, but may also cause tissue injury through excessive inflammation. Reactive oxygen species (ROS) are not only directly ore indirectly involved in a wide variety of clinical disorders, such as atherosclerosis, reperfusion injury, pulmonary toxicity and cancer, but they are also important in the aging process. This process is associated with increasing susceptibility to infection. In this study we investigated the influence of age and sex on phagocyte activation by means of a whole blood chemiluminescence (CL) assay. Circulating phagocyte activity was measured in 55 healthy volunteers (24 females, 31 males) aged from 6 to 92 years. Using an automated luminescence system, phagocytes were stimulated by polystyrene beads and Luminol-enhanced CL was determined in terms of peak height and peak time in freshly withdrawn, peripheral venous whole blood. An extremely significant positive correlation (p < 0.0001) between the maximum of light emission after stimulation and increasing age was found. This finding is true for the total population of blood phagocytes as well as for a single cell. In contrast the time of the appearance of the maximum of light emission showed an extremely significant inverse correlation (p < 0.0003) with increasing age. The influence of sex on the CL-parameters showed no significant difference between women and men. It is concluded that the increased susceptibility of circulating phagocytes to oxidative burst in elderly subjects may be the consequence of several biological events. Senescent cells express more and also have new antigens on their surfaces that trigger an autoimmune response. Cellular senescence appears earlier in old organisms. Therefore phagocytes in aging individuals may be increasingly involved in their scavenger tasks that grow with the catabolic bias in cell turnover. Moreover, atherosclerotic alterations in the intima and endothelial lesions are physiologic concomitants of age and may lead to a stimulation of circulating phagocytes.

Effect of oxidative stress by iron on 4-hydroxynonenal formation and proliferative activity in hepatomas of different degrees of differentiation.

It has been shown previously that oxidative stress by ferrous iron in vitro leads to an inhibition of proliferation of murine ascites tumour cells in vivo. This effect is associated with increased lipid peroxidation in terms of formation of the highly reactive aldehyde 4-hydroxynonenal (HNE), which has been shown to inhibit the proliferation of numerous tumours and to induce differentiation. It was the purpose of this article to study the occurrence and metabolism of HNE and its inducibility by oxidative stress in hepatomas of different degrees of differentiation to find further evidence for a possible role of HNE in proliferation and/or differentiation, because it is known that in hepatoma cells with a very low degree of differentiation basal lipid peroxidation is hardly detectable, while in normal hepatocytes the basal level of thiobarbituric acid reactive substances (TBArS) is rather high. MH1C1 hepatoma cells and Yoshida AH-130 hepatoma cells were chosen as highly differentiated and poorly differentiated tumour cells, respectively, and rat hepatocytes served as a control for normal liver phenotype. Ferrous histidinate (Fe/His) did not have a cytotoxic effect on Yoshida and MH1C1 cells, as measured by the LDH release test. In cell culture studies Fe/His revealed a dose dependent inhibition of the proliferation of Yoshida cells. The incorporation of 3H-thymidine into DNA of these cells was also inhibited by Fe/His in a dose-dependent manner, while the precursor uptake into the cytoplasm was unaffected. The basal levels of HNE were in the order: hepatocytes > MH1C1 cells > Yoshida cells. Both hepatocytes and Yoshida cells responded to the presence of Fe/His with increased formation of TBArS. Compared with hepatocytes the response of the Yoshida cells was greatly reduced. The response of cells to Fe/His with respect to HNE formation was decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells, but in this case the differences were not very pronounced. The metabolic capacity of the cells to consume HNE was also decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells. In this case the differences were very pronounced. These findings support the view that Yoshida cells with a low degree of differentiation and a low basal level of HNE are released from an inhibitory effect of HNE operative in hepatocytes and that HNE is causally involved in the iron induced inhibition of proliferation of poorly differentiated hepatoma cells.

Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4-hydroxy-2-nonenal.

The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE). GSH was entirely depleted within a 2 h incubation with 250 microM HNE. After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level. In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells. We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions. Furthermore, we have discovered a new indication for the stress marker trehalose in S. cerevisiae.

Human low density lipoprotein as a target of hypochlorite generated by myeloperoxidase.

The aim of this study was to further clarify which part of human low density lipoprotein (LDL) is attacked by the MPO/H2O2/Cl- -system and which reactive oxygen species is responsible for the attack. Therefore the influence of this system on the modification of the lipid and protein moiety of LDL was studied in vitro. Using the monochlorodimedone assay it was found that HOCl is produced in micromolar quantities in the absence of LDL and is rapidly consumed by LDL in a concentration dependent manner. The consumption of HOCl was reflected in the formation of HOCl-specific epitopes on apo B-100 as determined by an antibody raised against HOCl-modified LDL. The absorbency at 234 nm was applied to measure continuously the extent of modification of LDL. The general kinetic pattern of the absorbency measurement consisted of a lag phase where no LDL modification was observed, followed by a rapid increase of absorbency and a plateau phase. Finally the absorbency decreased due to LDL precipitation. Time dependent absorption spectra indicated that this kinetic pattern is mainly caused by light scattering due to particle aggregation rather than by a specific absorption at 234 nm due to conjugated diene formation. In agreement with this finding a low rate of thiobarbituric acid reactive substances (TBArS) formation was observed after a lag phase. The aggregation of LDL occurs most likely by modification of apo B-100, which was determined fluorimetrically in terms of LDL-tryptophan destruction in presence of the MPO/H2O2/Cl(-)-system. The kinetic course of tryptophan fluorescence generally consisted of a rapid decrease leveling off into a low plateau phase. Gas chromatographic determinations of linoleic acid in LDL in presence of the MPO system showed that this polyunsaturated fatty acid (PUFA) is easily attacked by HOCl. Consistent with this finding NMR spectra of HOCl modified LDL indicated a complete disappearance of bis-allylic methylene groups. Since lipid peroxidation products only partially account for this loss of PUFAs, other reactions of HOCl with unsaturated lipids--probably chlorohydrin formation--must be involved. Summarizing, although the rate of lipid peroxidation is low, both the lipid and the protein moiety of LDL are readily modified by the MPO system. It appears that the immediate consequence of apo B-100 modification is its aggregation. It is concluded that MPO, which has been detected in atherosclerotic lesions, is able to contribute to the modification of LDL into a form recognizable for uncontrolled uptake by macrophages.

Tumor host relations. I. Increased plasma cortisol in tumor-bearing humans compared with patients with benign surgical diseases.

Plasma cortisol was measured by a protein binding technique in 81 patients with malignant tumors of different extent and various sites and in 82 patients with benign surgical diseases. The mean value of the tumor patients (x +/- s = 165 +/- 69 micrograms cortisol/l plasma) was increased significantly compared with the benign surgical disorders (100 +/- 45 micrograms/l). Within the group of patients with benign surgical disorders there was little variation by the type of disease (cortisol mean values given in brackets): benign breast tumors (95), gall stones (107), ulcer of the stomach and duodenum (96), hernia (78), appendicitis acuta (112), and struma (90). The results are in accordance with the hypothesis that glucocorticoids are involved in the increased protein catabolism of skeletal muscles and other signs of cachectic tumor patients.

Tumor host relations. II. Influence of tumor extent and tumor site on plasma cortisol of patients with malignant diseases.

The increase of plasma cortisol in patients with tumors of five different sites compared with a control group of patients with benign surgical diseases amounted to: +39% (breast), +34% (stomach), +86% (intestine), +60% (skin) and +194% (gall bladder). The first detectable increase of cortisol occurred in patients with tumors classified T 2 according to the TNM scheme (+27% above the control). Highly significant increases were observed for T 3 (+82%) and T 4 (+77%) patients. Patients with palpable lymph nodes showed a most significantly increased cortisol mean value compared with patients without palpable lymph nodes. Similarly, the cortisol mean value of patients with distant metastases was significantly higher than the corresponding value of tumor patients without distant metastases. The question remains open, whether the primary site, the extent of the tumor or the occurrence of metastases is the main determinant for the cortisol increase.

Iron-induced lipid peroxidation and inhibition of proliferation in Ehrlich ascites tumor cells.

The purpose of this study was to find further experimental evidence for the postulated negative association between the extent of lipid peroxidation in tumor cells and their proliferative behavior. After incubation of Ehrlich ascites tumor cells at 37 degrees C for 30 min with increasing concentrations of Fe(II) histidinate (Fe/His) the following parameters were determined: the formation of lipid hydroperoxides was measured fluorimetrically after reaction with dichlorofluorescein; 4-hydroxynonenal was determined by reversed-phase high-pressure chromatography after derivatization with dinitrophenylhydrazine; as a third parameter of lipid peroxidation the formation of 2-thiobarbituric-acid-reactive substances was determined. The proliferative activity was determined by measuring the growth rate in vivo after reimplantation i.p. of the tumor cells into mice. Trypan-blue exclusion tests for viability were performed before reimplantation. The reliability of the trypan-blue exclusion tests was checked by comparing the results with another parameter of viability, the release of the cytosolic enzyme lactate dehydrogenase. The concentration both of lipid hydroperoxides and of 2-thiobarbituric-acid-reactive substances showed a biphasic dependence on the concentration of Fe/His with maximal increase at iron concentrations of 0.25 mM and 0.1 mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/10(9) cells) with increasing iron concentration in the range from 0.1 mM to 0.6 mM. The total number of tumor cells, when determined 5 days after reimplantation, continuously decreased with increasing iron concentration, showing half-maximal inhibition at about 0.22 mM Fe. The exclusion of the trypan-blue dye was unaffected by the presence of iron at any concentration used. Similarly, iron had no influence on the release of lactate dehydrogenase. The results support the hypothesis that 4-hydroxynonenal may act as an inhibiting messenger between endogenic lipid peroxidation and proliferation.

Tumor host relations. IV. Tissue distribution of citric acid cycle intermediates and of glutamate in tumor-bearing rats high level of alpha-ketoglutarate in solid Yoshida sarcomas.

To elucidate the origin of increased concentrations of alpha-ketoglutarate (KG) in tumor bearers the tissue distribution of KG together with the related metabolites citrate, succinate, malate, and glutamate was determined in tumor, liver, gastrocnemius muscle, and blood of rats bearing the solid Yoshida sarcoma and of tumor-free rats. The sum of these metabolites was significantly increased in host liver and blood, respectively, compared with the corresponding tissues of normal rats. Among single metabolites glutamate and malate were significantly increased in host liver. The absolute concentrations were highest in host liver with the exception of KG, which was highest in the tumor. This was taken as indicative for the tumor as the prime source of increased KG in blood of tumor-bearers. No significant metabolic deviations were found in gastrocnemius muscle. The concentration of KG in this muscle of both normal and host animals correlated significantly with that of glutamate. In the tumor the concentration of KG correlated significantly with that of citrate plus succinate plus malate. This type of correlation was absent in liver and muscle of both normal and host animals. Moreover, no correlation existed between KG and glutamate either in liver or in tumor. It was suggested that the metabolic flux through the citric cycle determines the concentration of KG in the tumor.

Tumor host relations. V. Nitrogen metabolism in Yoshida sarcoma-bearing rats. Reduction of growth rate and increase of survival time by administration of physiological doses of branched-chain amino acids.

The continuous administration of physiological doses of the branched-chain amino acids leucine, isoleucine, and valine (Leu-Ile-Val) to Yoshida sarcoma-bearing rats caused a significant increase in the survival time by 32% and a significant reduction of tumor size after 3 weeks of growth by 33%. The shift of the nitrogen balance to negative values during the cachectic stage was delayed but not prevented. On the average, less nitrogen (-47 mg/day) were lost by Leu-Ile-Val treated rats compared with untreated tumor-bearing animals (-91 mg N/day). It appeared that Leu-Ile-Val increased the synthesis of carcass proteins, while it left the proteolysis rate unchanged, since the excretion of urea and creatinine was unaffected by these amino acids. The daily excretion of alpha-ketoglutarate, which is correlated with tumor size during the early stage of growth, was decreased during the first 2 weeks by Leu-Ile-Val, but remained for a longer period on a high level than in untreated tumor bearers. The results point to an improvement of the metabolic resistance against carcass protein depletion of the tumor-bearing host by the administration of branched-chain amino acids.